Apparatus for analysing continuously discrete biological liquid samples

ABSTRACT

Analysis apparatus primarily for carrying out biological analyses using radioimmunoassay techniques includes a turntable disc for carrying a plurality of specimens. A delivery arm delivers successive samples to a row of dilution containers and simultaneously supplies a diluent. A transfer arm controlled by a code on the turntable disc picks a selected diluted sample and transfers it to one of a long series of incubation chambers each of which contains a reagent. A second reagent is added at the time of transfer and the mixture is incubated prior to removal by an appropriate device. After removal the active constituents are filtered and measured quantitatively by a counter. The pumps for transfer of liquids are peristaltic and where a sample has to be drawn up incorporate an integral bellows.

United States Patent Bagshawe et al.

l l APPARATUS FOR ANALYSING CONTINUOUSLY DISCRETE BIOLOGICAL LIQUID SAMPLES [75] Inventors: Kenneth D. Bagshawe, London;

' Derek N. Marchant, Hassocks, both of England [73] Assignee: The Hayward Foundation, London,

England [22] Filed: Feb. 24, 1971 2: Appl. No.: 118,426

[30] Foreign Application Priority Data Mar. 3, 1970 Great Britain 10,023/70 [52] US. Cl 250/106 SC [51] Int. Cl. G0lt 1/16 [58] Field of Search 250/715 R, 106 SC;

[56] References Cited UNITED STATES PATENTS 3,614,434 lO/l97l Horwitz ..25O/7l.5R

[4 1 Jan. 8, 1974 Primary Examiner-James W. Lawrence Assistant ExaminerDavis L. Willis Att0rneyPennie, Edmonds, Morton, Taylor & Adams [57] ABSTRACT Analysis apparatus primarily for carrying out biological analyses using radioimmunoassay techniques includes a turntable disc for carrying a plurality of specimens. A delivery arm delivers successive samples to a row of dilution containers and simultaneously supplies a diluent. A transfer arm controlled by a code on the turntable disc, picks a selected diluted sample and transfers it to one of a long series of incubation chambers each of which contains a reagent. A second rea gent is added at the time of transfer and the mixture is incubated prior to removal by an appropriate device. After removal the active constituents are filtered and measured quantitatively by a counter.

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INVENTORS Kumem llBnssuawE. bezel: N. "\aRcuANT BY; w/Zw, J76 8 4 ATTORNEY I APPARATUS FOR ANALYSING CONTINUOUSLY DISCRETE BIOLOGICAL LIQUID SAMPLES The invention relates to an analysis method, apparatus and system.

Proposals have already been made for the analysis of biological samples on a continuous flow basis with a view to speeding up analytical procedures and with a view to reducing the unit cost of analysis. However, such previously proposed apparatus has met with difficulties owing, at least in part, to carry-over from one sample to the next within the communication passages of the apparatus.

Numerous analysis methods for biological samples are available of which radioimmunoassay has particular application where the sample includes, or may include hormones, enzymes or other substances produced by normal or cancerous cells. The technique of radioimmunoassay serves to measure accurately the quantity of a specific species of a protein and/or a specific species of a polypeptide in a sample, for example urine, blood or plasma, by reaction with specific anti-bodies produced in an animal in response to injections with the protein or polypeptide to be measured. Further, isotopically labelled protein or polypeptide of the specific species to be measured is added to the sample and this competes with the specific protein or polypeptide of the unknown sample for binding sites on the antibody. In order to measure the degree of binding with the antibodies it may be necessary to add a further antibody or a carrier particle. Separation of isotopically labelled protein or isotopically labelled polypeptide bound to an antibody from corresponding substances which are not bound can be effected by filtration, centrifuging or electrophoresis.

It is possible to measure the presence quantitatively of many proteins even in very low concentrations, and this reduces the quantity of costly antibodies required, but, where large numbers of tests are required, the time consumed in carrying out the tests is excessive and testing of large sections of the population by way of screen ing, becomes impossible.

According to the present invention, there is provided analysis apparatus comprising means for successively delivering discrete biological samples to successive containers at a station at which each sample is diluted to produce a plurality of diluted samples, means for diluting the samples at the dilution station, means for transferring a selected said diluted sample to an incubation container, an incubator for incubating the selected diluted sample in the container and analysis means for analysing components of the diluted incubated sample.

According to the present invention, there is further provided liquid analysis apparatus incorporating a plurality of remotely controlled peristaltic pumps for delivering and/or transferring quantities of liquid to and- /or from containers of the apparatus, at least one of the pumps incorporating bellows means, operable at the end of each delivery cycle of the pump to produce a suck-back action preventing the formation of drips.

An embodiment of analysis apparatus in accordance with the invention will now be described, by way of example, with reference to the accompanying diagrammatic drawings, in which:

FIG. 1 is a perspective view of analysis apparatus in accordance with the invention;

FIG. 2 is a plan view ofa turntable for carrying original, undiluted samples and forming part of the apparatus shown in FIG. 1;

FIG. 3 is a side elevation of a device for disposing of unwanted diluted samples;

FIG. 4 is a plan view illustrating a specimen dilution arm and a sample selection arm;

FIG. 4A is a side elevation of the sample selection arm of FIG. 4;

FIG. 5 is a side elevation of a device for cleaning certain parts of the arms shown in FIG. 4;

FIG. 6 is a fragmentary plan view of an incubation container web and one of the pulleys on which it is mounted;

FIG. 7 is a side elevation of a device for capping the individual containers of the incubation container strip conveyor and part of the drive for the incubation con tainer web;

FIG. 8 is a vertical sectionthrough an incubator of the apparatus;

FIG. 9A is a vertical section of an arrangement for removing the contents of successive incubation containers;

FIG. 98 illustrates a detail of the arrangement of FIG. 9A;

FIG. 9C is a diagram of an associated peristaltic P p FIG. 10 is a diagram illustrating a pump used for certain fluid supply and withdrawal operations in the apparatus;

FIG. 11 is a section of the pump illustrated in FIG. 10;

FIG. 12 is a section of another pump used in the apparatus;

FIGS. 13A to 13C illustrate a device for adjusting the operation of one of the pumps; and

FIG. 14 is a circuit diagram of the control system of the apparatus.

In outline, the apparatus as illustrated in FIG. 1 includes a turntable disc or other carrier means which has two rings of apertures 101, 102 each containing a pot 103 or other specimen container which is preferably disposable but is, in any event, readily removable from the turntable disc. The turntable disc 100 has a plurality of sets of pegs I04 selectively engaged in holes, or notches, the rows of holes or notches extending radially inwardly from the rings of apertures 101, 102. The position of thepeg 104 in any given radial row dictates the required dilution or neat specimen from which a sample is taken. Each sample is withdrawn from its pot 103 by delivery means 109 and diluted to a series of dilutions in a row of depressions 110 or other dilution containers in a plastics band 111 and a selected diluted or undiluted sample is transferred by transfer means 112 to an incubation train which includes a row of disposable plastics containers 131. The incubation train is initially stored in a refrigerated compartment 140. One reagent is already contained in each container 131 and another is added preferably simultaneously with transfer to the container 131, and the biological reactions are allowed to proceed with the aid of thorough mixing by agitation at the time of transfer and for about three minutes thereafter. The incubation train then follows an elongate path through an incubator 150. The length of this path through the incubator and the speed of the incubation train is such that the elapsed time from the addition of the reagents to the separation of the constituent to be measured corresponds to the required incubation time (say 2 hours at 37 C), for the reagents and sample in question. In the embodiment disclosed, the incubation train is indexed forward at one minute intervals.

On completion of the incubation, the samples are removed from the containers 131 by an incubatedsample removal means 160 and the molecular complexes in the incubated material are separated into the antibody-bound and antibody-free phases either by filtration, centrifuging or electrophoresis as is conventional in radioimmunoassay techniques. In the preferred embodiment a vacuum filtration process is employed, the mixture being released on to a filter material by successively withdrawing the incubated sample from each container of the incubation train and washing out the whole of the contents on to the filter material by gelatin or other washing agent dispensed by the removal means 160.

The filter bed material (not shown) moves past or through an isotopic counter and the whole bed, including sample solids, is passed to waste. The output of the isotopic counter is supplied to a pulse-height analyser (not shown) which, in turn passes its output to a rate meter. If desired, the signals from the rate meter are recorded on a chart recorder.

Preferably the output of the multiplication stage is fed via a scaler to a data processor which correlates the result with sample identification information and the selected degree of dilution in order to produce comprehensive digital information at a final print-out.

For any one analysis apparatus more than one disc 100 and set of pots 103 will be provided, since, when one set of samples is being processed another set is being loaded with fresh sample-containing pots.

Provision may be made, as will be described hereinafter in detail, for continuing the incubation stage of the analysis after the part of the apparatus upstream of the incubator has been shut-down. Since the incubator requires no supervision, an extension of the working day of the apparatus can be achieved.

Certain parts of the analysis apparatus and system will now be described in more detail.

Referring particularly to FIG. 1, the specimens to be tested are arranged in the moulded plastics pots 103 which are lipped to enable them to engage the periphery of appropriately sized apertures 101, 102 in the turntable disc 100 which is rotatable about a vertical axis. The two annular rows 101, 102 of apertures are arranged with the individual apertures of one row 101 alternating in the circumferential sense with individual apertures of the other row 102. In other words, no one aperture has its centre line on the same radial line as another. Radially inwardly of each pot-receiving aper ture, there is a radially extending line of eight holes 105 (FIG. 2) of smaller diameter than the pot-receiving apertures. These smaller apertures 105 serve as a part of the control system of the apparatus, and each denotes a different degree of dilution required for the portion ofthe sample (or next specimen) to be deposited in the incubation train. It will be apparent that the radial rows of control apertures 105 also form eight annular rows and a micro-switch (not shown) is associated with and lies below each circular row so that a peg 104 inserted in one of the apertures 105 trips a micro-switch appropriate to the final degree of dilution (or non-dilution) selected and this in turn controls the operation of the transfer means 112 and pump hereinafter described in greater detail. The pegs 104 are held in place by a circular plate 106 (FIG. 1) and the disc as a whole is readily removable from the apparatus.

It will be noted that four of the receptacle-receiving apertures 101, 102 are omitted, one from the outer row and three from the inner row, and the corresponding control holes are also omitted. This facilitates starting and stopping a cycle of operation lnwardly of the holes 105, two further, inner, rings of dilution-control holes 105 are present for providing, for example, a 2:1 or 5:1 dilution of the sample and again pegs 104 can be inserted therein to provide the required dilution rate in co-operation with appropriate micro-switches (not shown). A horizontally extending flange 106A (broken lines) upon which the disc 100 is mounted by keying also actuates by means of an annular row of recesses 107, two indexing micro-switches 107. These position-control micro-switches 107' serve to control the indexing movements of the disc 100 through positions in which pots of the outer row 102 are available for sample delivery and on completion of pots of this row a switch 107" engages a recess 10613 and thus causes the flange to be indexed initially through one half of the pitch of the recesses 107 so that the other one of the switches 107' becomes operative. The flange is then indexed as before until the recess 1068 which actuates the switch 107" again actuates the switch to stop the apparatus when all the specimens have been dealt with.

The switch 107 also initiates a change in operation of the delivery and dilution arm, in that the transverse movement is extended to cover the inner row. The turntable disc 100 is driven intermittently by an electric motor (not shown) and is stopped exactly in each indexed position by a brake (likewise not shown) under control of the indexing switches 107. Alternatively if a synchronous motor is used, the brake can be omitted.

Details of the control system have been omitted from the general description but are illustrated by FIG. 14, however, at this stage attention is drawn to the timers arranged conveniently in a bank T and the relays in a bank R. A power supply input is indicated at S and a control panel C lies between the timer bank T and the turntable 100. A vacuum gauge V indicates the vacuum applied to the filter process in the analysis counter.

The turntable 100 lies adjacent the delivery means and the transfer means 109, 112, The band 111 is vacuum-formed to provide the continuous series of transverse rows of depressions 110, seven in each row. To reduce interruption in the operation of the machine, it is clearly desirable to have a supply of dilution containers in the band which will last for at least one day of continuous operation.

For a reason which will appear hereinafter a probe for delivering the original sample and successively diluted samples to the dilution containers is in the form ofa curved hypodermic type needle and because of the curvature there is a tendency for the liquid delivered to take on an undesirable component of motion which results in the liquid spilling over the rim of the container which is clearly inadmissible. To prevent this each dilution container (FIG. 3) is formed with a flat bottom A and a frustoconical side wall 1108. The side wall 110B extends at an angle of 92 r to the plane of the bottom 110A. Although increased manufacturing difficulty may result, even greater certainty that spillage 

1. In analysis apparatus means for successively delivering discrete biological specimens to successive dilution containers at a station at which each specimen is to be diluted, means for diluting the samples in the dilution containers at the dilution station to produce from each successive sample a plurality of samples each of different dilution, a series of incubation containers, means for transferring a sample of selected dilution to one of said incubation containers, an incubator for incubating the selected diluted sample in the said incubation container and analysis means for analysing the diluted sample.
 2. Apparatus according to claim 1, wherein the analysis means employs radioimmunoassay techniques.
 3. Apparatus according to claim 1, comprising incubated-sample removal means comprising a pump operative to remove the incubated sample material from successive said incubation containers, capable of delivering a succession of wash fluid quantities to each said container and successively removing said wash fluid quantities whereby all solids of the sample are removed for analysis by the analysis means.
 4. Apparatus according to claim 3, wherein the incubated sample removal means comprises a seal for sealing off each incubation container mouth, a probe for delivering air under pressure into the sealed container and an outlet through which the incubated sample and successive wash liquid quantities can be delivered to the analysis means.
 5. Apparatus according to claim 1, wherein the analysis apparatus includes a filter tape of glass fibre and a radiation counter.
 6. Apparatus according to claim 1, including circuit means for controlling and effecting operation of the component parts of the apparatus.
 7. Apparatus according to claim 1 incorporating a plurality of remotely-controlled peristaltic pump units for delivering and transferring quantities of liquid to and from containers of the apparatus, and bellows incorporated in at least one said unit operable at the end of each delivery cycle of the unit to produce a suck-back action preventing the formation of drips at a delivery terminal.
 8. Apparatus according to claim 7, comprising a diaphragm sealing off the interior of the bellows from the biological liquid.
 9. Apparatus according to claim 8, wherein the peristaltic pump includes means for maintaining the correct alignment of the flexible tube thereof and means for adjusting the amount of liquid delivered by each cycle of the pump.
 10. Apparatus according to claim 9, wherein the control means comprises a roller and means for adjusting its degree of contact with the tube at a position intermediate operational pinch rollers of the pump.
 11. Apparatus according to claim 7 comprising means for adjusting the timing of the active suction stroke of the bellows relatively to the peristaltic pump stroke.
 12. Apparatus according to claim 11, wherein the adjusting means comprises a first ring carrying a first contact, a second ring carrying a Second contact, a screwed arcuate member one end of which is secured to one of the rings and the other end is connected to the other ring, a projection on the other ring receiving the other end portion of the arcuate member and two nuts threaded on the arcuate member at opposite sides of the projection.
 13. Apparatus according to claim 1 wherein said diluting means comprises means identifying each said specimen and providing, in the form of a code, the appropriate degree of dilution required in the analysis, said transfer means being operable by the code to transfer the contents of a selected one of the dilution containers determined by one of the series of incubation containers.
 14. Apparatus according to claim 13, wherein the transfer means comprises an arm rotatable about its longitudinal axis and having a radial extension, and a probe carried on the radial extension, said arm being capable under the control of the identifying means of moving the probe into a selected one of the dilution containers and delivering the contents to one of the incubation containers.
 15. Apparatus according to claim 14 comprising drive means for the dilution containers including a wheel having radially extending pockets arranged to be engaged by the outside surfaces of the containers, and further comprising vibratory means arranged to impart vibration to the containers through an outer one of the longitudinal rows of containers whereby mixing of the diluent is accelerated.
 16. Apparatus according to claim 1 comprising refrigerator means for storing the incubation containers together with the reagent used in the analysis.
 17. Apparatus according to claim 1, comprising means for wiping surplus material from the transfer means after delivery of the selected diluted sample to the respective incubation container.
 18. Apparatus according to claim 17, wherein the surplus material wiping means comprises a twin layer strip of absorbent paper the layers of which are spaced apart with one layer lying above the other at least in the region of active wiping of the transfer means, and means for indexing the strip in synchronism with movements of the transfer means.
 19. Apparatus according to claim 1, comprising a disc rotatable about a vertical axis with two rows of peripheral apertures carrying specimen containers, the said disc incorporating inwardly of the peripheral apertures, sample identifying means in the form of pegs selectively inserted in one or more of a plurality of holes in the disc arranged in radial rows, each row being associated with a corresponding container aperture and substantially aligned with one of the undiluted specimen-container receiving apertures, and switch means, operable by said pegs, which switch means in turn activate the transfer means.
 20. Apparatus according to claim 1, wherein the means for delivering the undiluted specimen to each one of the plurality of dilution containers comprises a cam-controlled arm, a probe mounted on the arm and operable to dip into said undiluted specimen container, to pick up the sample, and successively to deliver the specimen into successive diluted-sample containers, and simultaneously to increase progressively the dilution in said successive dilution containers.
 21. Apparatus according to claim 1 comprising a web mounting the incubation containers, the mouths of the containers lying in a plane substantially at right angles to the plane of the web.
 22. Apparatus according to claim 21, wherein the incubator comprises means defining a substantially closed chamber, a plurality of pulleys within the chamber around which the incubation container web is trained, and means biasing at least one of said pulleys in a direction normal to the rotational axis of the pulley whereby the tension in the web is maintained substantially constant.
 23. Apparatus according to claim 21, wherein said incubator comprisEs means defining a chamber in which the incubation web is mounted, means defining a mixing chamber lying below the incubation web chamber, a tangential flow fan for circulating warm air through the two chambers, and a heater element in the mixing chamber downstream of the fan outlet.
 24. Apparatus according to claim 1, comprising means for placing an adhesive cap over the mouth of each successive filled incubation container whereby the evaporation rate is substantially reduced.
 25. Apparatus according to claim 1 comprising a device for flushing a said incubation container of a solids-containing liquid so that substantially no solids deposited by the liquid are left in the container, said device comprising a head, a seal carried by the head and capable of sealing off the container at the mouth thereof, a probe mounted in the head of a length such that the tip thereof can extend substantially to the bottom of the container, means defining an inlet in the head for delivering gas under pressure to the container, and an inlet for the delivery of wash liquid, a peristaltic pump for delivery in timed sequence pressurized gas to the gas inlet and wash liquid to the wash liquid inlet, and means for cycling the gas and liquid through a plurality of pressurization and wash liquid cycles.
 26. Apparatus for carrying out continuous discrete analysis of biological specimens using the radioimmunoassay technique, said apparatus comprising a turntable disc carrying a plurality of undiluted specimen containers, means on the disc associated with each container for indicating the required degree of dilution of the contents of each container, an undiluted-specimen delivery arm operable to withdraw successively the specimens from the undiluted-specimen containers, a plastics band formed with a row of dilution containers for receiving the withdrawn specimens, means for progressively increasing the degree of dilution in the successive dilution containers of the row, means for indexing the band after a row of containers has been filled with diluted samples of a given specimen, a series of incubation chambers arranged as a single row and connected to a flexible web, a diluted sample transfer arm actuable in dependence upon the indicating means to transfer a sample of selected dilution ratio to one of the incubation containers, an incubator incorporating means for recirculating continuously a heated air flow and means for indexing the incubation web through the incubator, an analysis apparatus, an incubated sample removal device for removing successive incubated samples to the analysis apparatus, said analysis apparatus including a glass fibre tape on which the solids in the incubated samples are retained, and a radiation counter for counting the radio-active material in said solids, peristaltic pump units coupled respectively to the specimen delivery arm, the sample transfer arm, and the incubated sample removal device, electric motors for driving respectively the delivery arm, the transfer arm, and the incubation container web and timing means for controlling operation of the motors. 